Long-term goal: Statins selectively inhibit the enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase leading to decreased cholesterol biosynthesis. Several natural and synthetic statins enhanced apoptosis in human lymphoblastoid, myeloma and breast cancer cells. This effect was directly related to their ability to inhibit HMG CoA reductase, which blocks the synthesis of isoprenylated small GTPases, and not by squalene, an immediate precursor of cholesterol. This proposal is based on the discovery that statins cause increased cytotoxicity to breast cancer cells through either increased expression of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) and/or decreased arginase expression. Statin-mediated cell death was partially reversed by 1400W, a more specific inhibitor of iNOS (NOS II), and by mevalonate, an immediate metabolic product of acetyl CoA/HMG-CoA reductase reaction. Mevalonate supplementation inhibited statin- induced iNOS and NO and restored arginase expression. Fluvastatin dose-dependently inhibited mammary tumor development in an in vivo animal model. Hypotheses to be tested are: (i) statins stimulate NO in breast cancer cells that is responsible for their proapoptotic, tumoricidal and antiproliferative effects, (ii) statins inhibit arginase expression and activity through inhibition of RhoA signaling in breast cancer cells, and (iii) supplementation with sepiapterin (iNOS co-factor) and tocotrienols potentiates statin-induced tumoricidal effects in breast cancer cells and in a rat model. Specific aims: (i) Assess the effects of various statins (lipophilic and hydrophilic) and tocotrienols on breast cancer cell proliferation, and apoptosis, (ii) Determine the induction of iNOS and NO formation in cells treated with statins alone and with sepiapterin and arginase inhibitors, (iii) Define the role of RhoA in statin-mediated NO generation, arginase expression, Nf:B inhibition and antiproliferative effects in breast cancer cells, (iv) Establish a chemopreventive rat model, and evaluate the effectiveness of statins alone and in combination with 3-tocotrienol or sepiapterin. Methods: We will use MCF- 7 and MDA-MB-231 cells and a chemically-induced breast cancer rat model. HPLC techniques will be used to detect and quantitate NO formation in cells treated with statins. Magnetic resonance imaging (MRI) will be used to assess the response to breast cancer therapy in a rat model. Significance: Recent research suggests that statins may prevent various types of cancers including breast cancer. However, the molecular mechanisms by which statins induce breast cancer cell death remain unknown. This proposal will advance our understanding of the chemopreventive and chemotherapeutic ability of statins, alone and in combination with naturally-occurring tocotrienols. Novelty: The overall goal is to elucidate the molecular mechanism by which statins exert antiproliferative/proapoptotic effects in breast cancer cells. The use of tocotrienols to synergistically enhance chemopreventive efficacy of statin in breast cancer cells and breast cancer animal model is innovative. MRI will be used to monitor chemopreventive effects of breast cancer in a rat model. PUBLIC HEARLTH RELEVANCE: Statins are one of the most widely prescribed group of drugs. Recent studies suggest that lipophilic statins may be beneficial for postmenopausal women. Studies also suggest that statins, when combined with other nutrients, become more potent as anticancer drugs. Breast cancer is the leading cause of death in women. Thus, it is both timely and important to understand the mechanism(s) by which statins kill breast cancer cells and to explore the possibility for clinical implementation of statins as chemopreventive drugs.